Project Summary/Abstract Glucocorticoid-induced leucine zipper (GILZ) is a broadly-expressed transcription factor whose expression is induced via endogenous or synthetic glucocorticoid (GC) signaling through the glucocorticoid receptor (GR). GILZ is thought to meditate many of GCs? anti-inflammatory effects in T lymphocytes, such as induction of TGF? signaling and inhibition of NF?B, MAPK, and AP-1 pathways. Conditional deletion of GILZ in CD4+ T cells leads to enhancement of experimental dinitrobenzene sulfonic acid (DNBS)-induced colitis, a Th1-mediated form of experimental inflammatory bowel disease (IBD). Steroid hormones, including corticosteroids and sex steroid hormones such as 17?-estradiol (estrogen, E2), demonstrate considerable promiscuity in receptor binding. E2 has been shown to antagonize GILZ in human uterine epithelial cells, but little is known regarding the mechanism(s) by which it may contribute to GILZ expression and/or function in T cells. Our preliminary data reveal a robust induction of GILZ at the mRNA and protein levels in regulatory T cells (Tregs) isolated from Crohn?s disease (CD) patients and SAMP/YitFC (?SAMP?) mice, a spontaneous model of Crohn?s-like disease. GILZ upregulation (?GILZhigh?) in these Tregs was associated with a relative decrease in estrogen receptor beta (ER?) expression, suggesting that GILZ expression in Tregs may be responsive to estrogen signaling. Furthermore, GILZhigh Tregs exhibited reduced suppressive function compared to their GILZlow counterparts, suggesting that optimal GILZ-mediated immunosuppression in Tregs may require intact ER? signaling. Several chronic inflammatory and autoimmune diseases exhibit reductions in ER? expression and/or activity, leading to the intriguing possibility that diminished ER? expression contributes to inflammation via disruption of normally-protective Treg GILZ-mediated mechanisms. Our hypothesis is that the protective functions of Treg- specific GILZ require intact ER? signaling, and therefore fail to mediated sufficient immunoprotection in ER?-depleted environments, such as the IBD intestine. The goal of this project is to determine the mechanism(s) by which reduced ER?-specific signaling influences the expression and function of GILZ in IBD- associated Tregs. We will make use of novel tools (GILZ transgenic and knockout mice and MaxCyte lentiviral transfection of primary human and murine T cells) to manipulate GILZ expression in ER?-deficient versus ? sufficient Tregs in order to understand how intact ER? signaling influences the expression (Subaim 1a) and function (Subaim 1b) of Treg-specific GILZ. Leveraging a large cohort of CD patients available through the Cleveland Digestive Disease Research Core Center (DDRCC), we will apply single-cell RNA sequencing to CD patient mucosal Tregs (discarded surgical samples) in order to identify unique transcriptional signatures (Subaim 1c) associated with ER?low/GILZhigh Tregs. These assays will provide critical data to inform future proposals focused on the intersection of E2 signaling and GILZ activity in Tregs. Successful completion of this proposal will provide mechanistic insight into the regulation of Treg GILZ expression and function via estrogen signaling. These studies will determine the link between diminished ER? signaling, characteristic of many auto- inflammatory diseases including IBD, and glucocorticoid signaling, with an ultimate goal of developing improved personalized medicine approaches for CD patients.